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      Effects of Extracts from Thai Piperaceae Plants against Infection with Toxoplasma gondii

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          Abstract

          Herbal medicines and natural herb extracts are widely used as alternative treatments for various parasitic diseases, and such extracts may also have potential to decrease the side effects of the standard regimen drugs used to treat toxoplasmosis (sulfadiazine-pyrimethamine combination). We evaluated how effective the Thai piperaceae plants Piper betle, P. nigrum and P. sarmentosum are against Toxoplasma gondii infection in vitro and in vivo. Individually, we extracted the piperaceae plants with ethanol, passed them through a rotary evaporator and then lyophilized them to obtain crude extracts for each one. The in vitro study indicated that the P. betle extract was the most effective extract at inhibiting parasite growth in HFF cells (IC 50 on RH-GFP: 23.2 μg/mL, IC 50 on PLK-GFP: 21.4 μg/mL). Furthermore, treatment of experimental mice with the P. betle extract for 7 days after infection with 1,000 tachyzoites of the T. gondii PLK strain increased their survival (survival rates: 100% in 400 mg/kg-treated, 83.3% in 100 mg/kg-treated, 33.3% in 25 mg/kg-treated, 33.3% in untreated mice). Furthermore, treatment with 400 mg/kg of the P. betle extract resulted in 100% mouse survival following infection with 100,000 tachyzoites. The present study shows that P. betle extract has the potential to act as a medical plant for the treatment of toxoplasmosis.

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          Toxoplasmosis: A history of clinical observations.

          It has been 100 years since Toxoplasma gondii was initially described in Tunis by Nicolle and Manceaux (1908) in the tissues of the gundi (Ctenodoactylus gundi) and in Brazil by Splendore (1908) in the tissues of a rabbit. Toxoplasma gondii is a ubiquitous, Apicomplexan parasite of warm-blooded animals that can cause several clinical syndromes including encephalitis, chorioretinitis, congenital infection and neonatal mortality. Fifteen years after the description of T. gondii by Nicolle and Manceaux a fatal case of toxoplasmosis in a child was reported by Janků. In 1939 Wolf, Cowen and Paige were the first to conclusively identify T. gondii as a cause of human disease. This review examines the clinical manifestations of infection with T. gondii and the history of the discovery of these manifestations.
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            Management of Toxoplasma gondii infection during pregnancy.

            Acute infection with Toxoplasma gondii during pregnancy and its potentially tragic outcome for the fetus and newborn continue to occur in the United States, as well as worldwide, despite the fact that it can be prevented. The infection can be acquired through ingestion of infected, undercooked meat or contaminated food or water. Transmission to the fetus occurs almost solely in women who acquire their primary infection during gestation and can result in visual and hearing loss, mental and psychomotor retardation, seizures, hematological abnormalities, hepatosplenomegaly, or death. Systematic education and serological screening of pregnant women are the most reliable and currently available strategies for the prevention, diagnosis, and early treatment of the infection in the offspring; this is largely because toxoplasmosis in pregnant women most often goes unrecognized. Treatment of the infection in the fetus and infant during the first year of life has been demonstrated to significantly improve the clinical outcome.
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              Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection

              Background Worldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences. Methods To better understand long-term effects and pathogenesis of this common, persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5–12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or αPD1 ligand were studied. Results Twelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimer's disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation. Conclusion In outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                23 May 2016
                2016
                : 11
                : 5
                : e0156116
                Affiliations
                [1 ]National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080–8555, Japan
                [2 ]Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, 999 Phutthamonthon Sai 4 Road Salaya, Phutthamonthon Nakhonpathom 73170, Thailand
                Ehime University, JAPAN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AL YN. Performed the experiments: AL SB NS YN. Analyzed the data: AL YN. Contributed reagents/materials/analysis tools: AL SB NS. Wrote the paper: AL YN.

                Article
                PONE-D-16-02479
                10.1371/journal.pone.0156116
                4877092
                27213575
                2bfca493-f897-434d-b644-9a50c9358197
                © 2016 Leesombun et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 January 2016
                : 8 May 2016
                Page count
                Figures: 5, Tables: 0, Pages: 13
                Funding
                Funded by: Obihiro University of Agriculture and Veterinary Medicine
                Award Recipient :
                The authors received no specific funding for this work.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Protozoans
                Parasitic Protozoans
                Toxoplasma
                Toxoplasma Gondii
                Medicine and Health Sciences
                Parasitic Diseases
                Biology and Life Sciences
                Parasitology
                Parasite Groups
                Apicomplexa
                Tachyzoites
                Research and Analysis Methods
                Biological Cultures
                Cell Lines
                Vero Cells
                Medicine and Health Sciences
                Parasitic Diseases
                Protozoan Infections
                Medicine and Health Sciences
                Parasitic Diseases
                Protozoan Infections
                Toxoplasmosis
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Alcohols
                Ethanol
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Alcohols
                Ethanol
                Research and Analysis Methods
                Mathematical and Statistical Techniques
                Statistical Methods
                Analysis of Variance
                Physical Sciences
                Mathematics
                Statistics (Mathematics)
                Statistical Methods
                Analysis of Variance
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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